Jun 28, 2021 High molecular weight bacterial DNA extraction from field-collected fecal samples preserved in ethanol for long-read sequencing In 1 collection Amandine Magdeleine , Marie-Ka Tilak , Sophie Teullet , Frédéric Delsuc Institut des Sciences de l’Evolution de Montpellier (ISEM), CNRS, IRD, Université de Montpellier, Mo ntpellier, France High molecular weight DNA extraction from all kingdoms Tech. support email: See@each.protocol See@each.protocol Sophie Teullet Institut des Sciences de l'Evolution de Montpellier 1 1 1 1 1 3 Works for me Share dx.doi.org/10.17504/protocols.io.bvyin7ue ABSTRACT Long-read metagenomic sequencing enables the reconstruction of better-quality assemblies compared to short-read assemblies. Long-read assemblies are typically more contiguous and this is of interest to recover full bacterial genomes. Here, we propose a protocol to extract high molecular weight bacterial DNA from mammalian fecal samples for long-read sequencing on the portable MinION instrument. DNA was extracted from feces collected in the field or from roadkill specimens and preserved in ethanol (96%) and stored at -20°C. The DNA extraction was done according to the “Genomic DNA from soil” kit (NucleoSpin Soil, Macherey-Nagel) which was optimized in three ways: i) SL1 + SX lysis buffers were used because this combination gave a high yield and degraded DNA the less, ii) two successive extractions were performed to get long fragments of DNA and iii) an additional DNA sizing step was added after the extraction to remove the remaining short DNA fragments and ensure DNA purity. The comparison of the two successive extractions showed that the obtained reads did not differ in terms of taxonomy but only the second yielded high molecular weight DNA suitable for long-read sequencing. Resulting long-read assemblies were more contiguous than Illumina short-read ones with fewer contigs (~4 000 and ~50 000 respectively) and a higher N50 (~90 000 kb and ~10 000 kb respectively) illustrating the advantage of using long reads to reconstruct bacterial genomes from ethanol- preserved fecal samples. DOI dx.doi.org/10.17504/protocols.io.bvyin7ue 1 Citation: Citation: Amandine Magdeleine, Marie-Ka Tilak, Sophie Teullet, Frédéric Delsuc High molecular weight bacterial DNA extraction from field-collected fecal samples preserved in ethanol for long-read sequencing https://dx.doi.org/10.17504/protocols.io.bvyin7ue This is an open access protocol distributed under the terms of the Creative Commons Attribution License Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,